The overall goal of this project is to clone the gene for cystic fibrosis. If this can be accomplished, additional goals would be to explore the possibility of a heterozygote test suitable for use in the general population, and to explore any implications for therapy. A genomic DNA library has been prepared from a Chinese hamster cell hybrid which contains a small portion of human chromosome 7, highly likely to include the CF locus. Overlapping cosmids will be organized into contiguous DNA segments. Cosmids will also be grouped by mapping to pulsed field gel fragments. Genomic DNA regions from the cosmids will be evaluated using crossovers between linked markers and the CF locus, pulsed field gel electrophoresis and other strategies. Conserved and transcribed regions will be identified using Southern blotting with DNA from various species as well as Northern blotting analysis. Alternative strategies to clone the CF gene will be pursued using a concept of candidate gene families and strategies such as subtraction hybridization. For candidate genes identified using genomic DNA clones, cDNAs will be isolated and analyzed in attempts to prove whether they may or may not represent the CF gene product. CF cells will be analyzed by Southern blotting, by Northern blotting, by nuclease analysis of RNA and by nucleic acid sequencing to determine if a candidate gene is the CF gene. If the CF gene can be identified, efforts will be focused on development of a heterozygote test suitable for use in the general population. This could be based on a biological method or on a direct molecular analysis. In addition, longer term goals might focus on the potential for somatic gene replacement therapy using retroviral vectors encoding the CF gene product.